3-Metyladenine (3-MA) and rapamycin (Rapa) were obtained from Sigma-Aldrich (St. Louis, MO). For 3-MA treatment studies, cells were seeded and let adhere on sterile plastic dishes for 24h prior to start any treatment. After 24h, the cells were treated with 10mM 3-MA for further 4h. For rapamycin treatment studies, the cells were treated with 2.5μM rapamycin or DMSO (Invitrogen, Carlsbad, CA, USA) for further 48h after transfection of GapmeR AK156230 or GapmeR control. All experiments were carried out in triplicate.
Rapamycin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Rapamycin is a macrocyclic lactone compound produced by the bacterium Streptomyces hygroscopicus. It functions as an immunosuppressant and has been used in various medical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Fm4 64, by Thermo Fisher Scientific (6 mentions)
Chloroquine, by Merck Group (5 mentions)
Thapsigargin, by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Carbachol, by Thermo Fisher Scientific (4 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
3 methyladenine 3 ma, by Merck Group (3 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Metformin, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Tunicamycin, by Merck Group (3 mentions)
Bafilomycin a1, by Merck Group (3 mentions)
Rapamycin, by Merck Group (3 mentions)
Il 12, by Miltenyi Biotec (3 mentions)
Il 15, by Thermo Fisher Scientific (3 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
103 protocols using rapamycin
1
Evaluating 3-MA and Rapamycin Effects
2
Yeast Cell Growth and Membrane Imaging
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Cells were grown overnight in YPD or SD with the appropriate dropout to maintain plasmid selection. Cells were then diluted in SC and grown to mid–logarithmic phase. For the various nutrient conditions, cells were then washed and transferred to the appropriate medium for the indicated times. For plasma membrane colocalization studies, cells were placed on ice and incubated with 10 μM FM 4-64 (Thermo Fisher Scientific) for 30 min, followed by immediate imaging. For rapamycin treatment, cells in YPD were treated for the indicated time with a final concentration of 200 ng/ml rapamycin (Thermo Fisher Scientific). Cells were plated onto No. 1.5 glass-bottomed coverdishes (MatTek Corporation, Ashland, MA) previously treated with 15 μl 2 mg/ml concanavalin-A (Sigma-Aldrich, St. Louis, MO).
3
Cannabinoid and Anti-Aging Drug Effects on Fibroblasts
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Cell cultures were treated with THC (Cat# T4764, Sigma-Aldrich, Saint Louis, MO, USA) and CBD (Cat# C-045, Sigma-Aldrich, Saint Louis, MO, USA) at the following doses: 0.25 µM, 0.5 µM, 1 µM, 2 µM, 5 µM, 7.5 µM, and 10 µM. Cannabinoids were dissolved in dimethyl sulfoxide (DMSO), anhydrous (Cat# D12345, Life Technologies Corporation, Carlsbad, CA, USA). Following H2O2 exposure, cells were incubated with the designated treatment for 2 h daily for 5 days due to previous studies showing optimal benefits occurring after 1–2 h exposures [24 (link),25 (link)]. Subsequently, the media was replaced without any additional treatments. Healthy fibroblasts (not exposed to H2O2) were treated with cannabinoids, as described above. In addition, CCD-1135Sk dermal fibroblasts were treated with three popular anti-aging drugs, rapamycin, metformin, and triacetylresveratrol (TRSV) at concentrations previously shown to be efficacious in anti-aging studies: 5 µM of rapamycin (Thermo Fisher Scientific, Waltham, MA, USA), 500 µM of metformin (Cedarlane, Toronto, ON, Canada), and 10 µM TRSV (VWR, Radnor, PA, USA) [18 (link)]. All anti-aging drugs were dissolved in DMSO and then dissolved in the media before being applied (n = 3 for each condition) for 2 h daily for 5 days.
4
Reversible Cellular Rejuvenation via Rapamycin
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The hiPSCs were cultured on Matrigel growth factor-reduced basement membrane matrix (Corning) in mTeSR medium (STEMCELL Technologies). The cells were treated with rapamycin (200 nM–2 μM, Fisher Scientific) or DMSO for indicated periods and thereafter tested for reversion in the absence of rapamycin. To analyze the proteasome function, hiPSCs were treated with 100 nM MG132 (proteasomal inhibitor, Thermo Scientific; cat. no. 508339) for 24 h. Information regarding cell lines is given in the supplemental experimental procedures .
5
Rapamycin Longevity Assay in Flies
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Rapamycin (Fisher Scientific) was dissolved in DMSO and added to the food at a final concentration of 200μM. 4–7 days old flies were shifted to food containing either DMSO or DMSO and Rapamycin for 4 days before being shifted to 29°C for 3 days. Food with either DMSO or DMSO and Rapamycin was changed every 2 days.
6
Confocal Imaging of Protein Translocation
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A Nikon A1R-HD resonant scanning laser confocal microscope was used, mounted on a Nikon TiE inverted microscope stand. Images were collected through a 1.45 NA, plan apochromatic oil immersion objective (Nikon). Green (EGFP) and far red (iRFP713) fluorescence were co-excited using the 488 and 640 nm laser lines of a fiber-coupled LUN-V combiner, whereas red (mCherry) fluorescence was excited on a subsequent line scan to avoid cross talk, using the 561 nm laser line. Emission was collected using the following filters: green (505–550 nm), yellow-orange (570–620 nm) and far-red (650–850 nm). Confocal planes were collected with a pinhole of 1.2 Airy Units calculated for the far-red channel in resonant mode with 8 or 16 frame averaging. For time lapse imaging, up to 10 fields of cells were selected using the motorized stage and imaged every 30 s. Addition of rapamycin was accomplished by pipetting 0.5 ml of complete HEPES imaging media containing 5 µM rapamycin (Fisher Scientific BP2963–1) to the 2 ml already in the dish, achieving a final bath concentration of 1 µM.
7
Thapsigargin and Rapamycin Induced Cell Stress Response
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Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was from Invitrogen (Carlsbad, CA, USA). DMEM and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The Hoechst kit and Lyso-Tracker Red probe for acidic lysosome staining were from Beyotime (Haimen, Jiangsu, China). Anti-PERK, Anti-BiP, anti-p-eIF2α, anti-eIF2α, anti-cleaved-caspase 3, anti-LC3, anti-p-AKT, anti-p-p70S6K1, anti-p70S6K1, anti-p-4EBP1 and anti-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ATF-6, anti-p62 and anti-GAPDH were from Abcam (Cambridge, UK). The CHOP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were obtained from Sigma-Aldrich with the highest purity available.
8
Cell Culture Protocol for Glioblastoma and Embryonic Kidney
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F98 and C6 rat glioblastoma, human glioma U87 MG, and human embryonic kidney 293T cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA), while the human glioma cell lines U251 and SHG44 were purchased from Institute of Basic Medical of Science, Research Chinese Academy of Medical Sciences. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (HyClone, UT, USA) in a humidified atmosphere with 5% CO2 and 95% air at 37°C. Puromycin (Invitrogen, USA), rapamycin (Invitrogen), Chloroquine (CQ; Invitrogen) and 3-methyladenine (3-MA; Sigma–Aldrich, USA) were dissolved in DMSO prior to use.
9
Validating mTORC1 Signaling Assay
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In western blot assays, all of the antibodies were of high quality and were validated. The pp70S6K, p70S6K, p4EBP1, 4EBP1, and beta-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), which were widely used in mTORC1 signaling study. For cell culture, RPMI-1640 and Fetal Bovine Serum (FBS) for PBMC culture were purchased from Gibco Invitrogen (Carlsbad, CA, USA). mTOR signaling inhibitors rapamycin and Torin1 were from Invitrogen (Carlsbad, CA, USA). Other chemicals were of the highest purity available.
10
Optimizing Autophagy Induction in hBM-MSCs
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rapamycin (Rapa), a well-known inducer of autophagy, was used to activate the autophagy pathway. To optimize rapamycin (Invitrogen, USA) concentration, hBM-MSCs were cultured in 96-well plates (Orange Scientific, Belgium), and different rapamycin doses (10, 200, and 500 nM) were added and incubated for 24 hours in a standard cell-culturing incubator. The rapamycin-treated hBM-MSCs were named MSC-10 nM Rapa, MSC-200 nM Rapa, and MSC-500 nM Rapa according to the rapamycin dose applied. MSC-LC3, MSC-shRNA Cont, and MSC-shRNA 3 treated with the optimized rapamycin dose were named MSC-LC3-Rapa, MSC-shRNA Cont-Rapa and MSC-shRNA 3-Rapa, respectively. These rapamycin-treated hBM-MSCs were then subjected to western blot analysis.
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